Composition containing ultra-micronized palmitoyl-ethanolamide

ABSTRACT

The present invention relates to a composition for pharmaceutical or veterinary use, comprising palmitoylethanolamide. In particular, the present invention relates to a pharmaceutical composition for human or veterinary use, containing a therapeutically efficient amount of palmitoylethanolamide in the ultra-micronized form, wherein more than 90% by weight of palmitoylethanolamide has particle sizes lower than 6 microns, together with pharmaceutically acceptable excipients.

FIELD OF THE INVENTION

The present invention relates to a composition for pharmaceutical orveterinary use, comprising palmitoylethanolamide.

DESCRIPTION OF THE STATE OF THE ART

In recent years, the concept of “neuroimmunogenic inflammation” has beenwidely developed, and important progresses have been made inunderstanding the biological mechanisms behind this widespread type oftissue inflammation, which is primarily induced by the release of givensubstances by the terminals of primary sensory neurons. Furthermore, ithas been shown that small-diameter sensitive fibres participate in theneuroimmunogenic inflammation phenomenon, which fibres are responsive tocapsaicin—the plant vanilloid that is present in the red chili pepper—,and that given neuropeptides, which are released by the above-mentionednerve fibres—particularly, the Substance P (SP) and the CalcitoninGene-Related Peptide (CGRP)— represent the main peptides responsible forthe occurrence of neuroimmunogenic inflammation peripherally.

The possibility to regulate the excitability of the sensory—bothnociceptive and pruriceptive—neurons currently has a relevant andincreasing therapeutic importance in a wide number of diseases affectingthe tissues of peripheral organs, both in man and animals.

Then, the most recent researches have put into focus the role that aspecific family of receptors, called TRPV, and in particular thereceptor TRPV1—initially known as capsaicin receptor VR1—plays in theprocess of neurogenic inflammation, and in particular in the hyperalgicphenomena associated thereto.

From the clinical point of view, the outcomes of the new knowledge onthe neuroimmunogenic inflammation mechanisms result to be of greatinterest in the Irritable Bowel Syndrome, in the interstitial cistitis,in the vulvodynias and vestibulodynias, in the vulvar vestibulitis, andin the chronic abacterial prostatitis, in the endometrial lesions, inthe miastenia gravis, in the arthropathies of traumatic or degenerativeor immunologic origin, affecting the joints, in the painful diseases ofthe intervertebral discs due to neoinnervation and neovascolarization ofthe cartilaginous tissue and the annexed ligamentous structures, in thecephalalgic syndromes due to inflammation of the meningeal tissue, inthe inflammatory states of the mucous and mucocutaneous tissues of theoral cavity and the dental pulp, in the recurrent fevers withauto-inflammatory basis of PFAPA type, particularly, even if notexclusively, in the pediatric age, in the postherpetic neuralgia, in theadherential syndromes due to peritonitis and/or laparotomic and/orlaparoscopic surgical events. The perspectives given by the biomedicalresearch in recent years are of great interest, in relation toneuroimmunogenic inflammation, both acute and chronic, at the skinlevel, as well as to implications between neuroimmunogenic cutaneousinflammation to psychogenic stimuli, such as stress, that configure moreand more clearly a tight connection between brain and skin. This is ofgreat importance in planning innovative pharmacologic approaches in aseries of dermatites of a erythematous-squamous nature, in the human andveterinary field (atopic dermatitis, irritative contact dermatitis,allergic contact dermatitis) characterized by itch, burning, localirritation, cutaneous rash, etc., as well as in chronic inflammatorydiseases of the granulomatous type at the level of the dermo-epidermal,and more generally, the connective tissues.

The neuroinflammation at the level of the spinal cord nervous structuresis characterized by the activation and proliferation of the microglialcells, which are normally present at the spinal level in a quiescentstate; such activation, mainly induced by the chronic and/or neuropathicpain, concurs in a relevant manner to the amplification of the painstimuli deriving chronically from the Peripheral Nervous System, or dueto damages localized in the brain, as well as to the development ofneurodegeneration through the microglial release of inflammatorymediators, and particularly of the pro-inflammatory cytokine TNF-alpha,interleukin IL-1 beta, and NGF. The activation and proliferation processof the microglia at the level of the spinal cord further plays anextraordinarily important role in the determination of neuropathic painconsequent to damages to the same nervous structures of the spinal cord:in fact, the activated microglia maintains an intense cytokinecommunication with the spinal cord neurons. All of this is veryimportant in diseases that originate from distresses, primarily of thespinal cord, such as the medullary canal stenoses and the traumaticlesions from flexo-extension of the spine (whiplash injury), and indiseases that, although depending on encephalic neuronal damages, due tothe activation effect of cells that are present in the spinal cord (inparticular the microglia), induce the symptomatology characteristic ofhyperalgic pain (Central Pain Syndrome) and, in given situations, ofspasticity; in particular, these are phenomena that are related todiseases such as Amyotrophic Lateral Sclerosis (ALS), MultipleSclerosis, post-stroke situations, Parkinson's disease, and fibromyalgicsyndrome.

The neuroinflammation at the level of the brain neronal structures,today better defined as reactive glyosis, currently represents one ofthe most interesting topics for the Neurosciences: in particular, thecause-and-effect relationship between the presence of neuroinflammatoryprocesses and neuronal degenerative damage (neurodegenerazione) is moreand more clearly defined, with the observation that theneuroinflammation due to the activation and proliferation ofnon-neuronal cells, such as microglia and astrocytes that are present inthe brain, represents the true cause for the degenerative damageaffecting the neuron. Moreover, it is evident how the activation ofmicroglia and astrocytes is induced and amplified by pro-inflammatorysignals, also of autocrine provenience, such as the TNFα and IL1β. Theneuroinflammation has been recognized as being an important causalfactor in many degenerative and traumatic diseases affecting the CNS,such as Parkinson's disease, Alzheimer's disease, stroke, cranialtrauma.

A highly innovative approach in order to intervene pharmacologically ondiseases supported by tissue neuroimmunogenic inflammation, or spinalneuroinflammation, or neuroinflammation of the encephalic nervousstructures may consist in the modulation, by means of variousmechanisms, of the activation of the non-neuronal cells controlling theperipheral and central sensitization of the neuronal cells, withoutthereby having to necessarily act directly on the neuron.

Furthermore, it has to be considered, in particular, that a number ofnon-neuronal cells, belonging to the immune system such as, for example,the microglia, are capable of expressing the cannabinoid CB2 receptorwhen suitably activated. The endocannabinoid 2-arachidonoylglycerol(2-AG) has been recognized in recent years as the true endogencannabinoid CB2 receptor ligand, and therefore, as an endogen substancethat is capable of modulating the activating and proliferative responseof immune progenitor cells strictly related to the sensitizationprocesses of the peripheral and spinal neurons.

Therefore, the object of the present invention is to provide apharmaceutical composition for the treatment of diseases related toneurogenic inflammation or neuroinflammation, both at the level ofperipheral organs and centrally.

Such object is achieved by a composition containingpalmitoylethanolamide as defined in the annexed claims, the definitionsof which form an integral part of the present description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph illustrating the concentration of PEA in serum inanimals treated with PEA or ultra-micronized PEA according to theinvention, as a function of time;

FIG. 2 shows a graph illustrating the concentration of 2-AG(2-arachidonoylglycerol) in serum in animals treated with PEA or withultra-micronized PEA according to the invention, as a function of time;

FIG. 3 shows a graph of MDSC (Modulated Differential Scanningcalorimetry) of original. PEA;

FIG. 4 shows a graph of MDSC (Modulated Differential Scanningcalorimetry) of ultra-micronized PEA according to the invention;

FIG. 5 shows a graph of XRD (X-Ray Diffraction) of original PEA;

FIG. 6 shows a graph of XRD (X-Ray Diffraction) of ultra-micronized PEAaccording to the invention.

DESCRIPTION OF THE INVENTION

The pharmaceutical composition of the invention containspalmitoylethanolamide (PEA) in the ultra-micronized form, wherein morethan 90% by weight of palmitoylethanolamide has particle sizes lowerthan 6 microns.

It has been surprisingly noticed that such a composition, compared toknown compositions containing PEA in micronized form, is provided with ahigh ability to peripherally and centrally act towards inflammatorydiseases of the neurogenic or neuroinflammatory type.

Palmitoylethanolamide, a substance of lipidic nature, is hardlysubjected to micronization methods, due to the easiness with which ittends to form aggregates; furthermore, the micronization in mechanicalenergy mills tends to heat the particles to be crushed, and therefore itpromotes such aggregation phenomenon, in practice being in contrast withthe desired object to decrease the compound particle size to amicrometer level.

Previously, a micronization of the palmitoylethanolamide had beenobtained, as described in EP 1 207 870 B1. Although the micronized PEAhad, in the treatment of some specific diseases, improvedcharacteristics compared to the non-micronized one, there were noexpectation that an effect could be obtained also in neuroinflammatorydiseases of the type that is treated in the present invention, nor therewas a motivation to push the micronization beyond the threshold of theparticle size obtained, both because there were no particularjustifications, and because, considered the lipidic nature of thesubstance subjected to the micronization process, the hope for a successwith the conventional technologies was incredibly low. In the tests thatwere initially carried out, in fact, a trend of the product to generatewaxy agglomerates had been noticed, also due to the temperature rise forprolonged dwelling times of the product particles in the micronizationchamber, which are necessary to obtain a more efficient micronization.

Therefore, in spite of the existing prejudices in the field, the presentinventors have surprisingly found that, by operating with a fluid jetmicronization process (that will be hereinafter referred to as“ultra-micronization”), and by suitably modifying the parameters of suchprocess, it is possible to obtain a still more efficient micronization,i.e., a particle distribution of PEA with particle sizes that arestatistically lower than those obtainable with the conventionalmicronization methods.

The product obtained following ultra-micronization has been furthercharacterized in comparison to the original product by a) MDSC(Modulated Differential Scanning calorimetry), and b) XRD (X-RayDiffraction) with the aim to detect possible structural modificationsinduced by the ultra-micronization process. Surprisingly, the inventorshave found that the ultra-micronized product shows a MDSC and XRDprofile that is completely different from the original product, thusdemonstrating the appearance, after ultra-micronization, of a differentcrystalline structure with a higher energy content.

Still more surprisingly, the inventors have found that such new particlesize profile of the PEA, and such different crystalline structurecharacterized by a higher energy content, corresponds to anexponentially increased pharmacological activity compared to themicronized PEA described in EP 1 207 870 B1, in diseases related toneurogenic inflammation or neuroinflammation, therefore bothperipherally and centrally.

The ultra-micronization process of the present invention is carried outin a fluid jet plant (for example, the plant model Jetmill®) operatingwith a pressurized air jet “spiral technology” that is capable ofexploiting the kinetic energy—in place of the mechanical energy—to crushthe palmitoylethanolamide particles. Such pieces of equipment areconventional, therefore they will not be further described.

In the described plant there are no mobile parts, and the productremains within the crushing disc for a very short time; the fluidthreads that are generated within the micronization chamber allowaccelerating the particles so that they can reach particularly highspeeds, such as to generate a sufficient energy so that they are crushedthrough a very high number collisions with each other and, as thecurrent inventors found in the case the ultra-micronization process, toinduce the modifications of the crystalline structure with theappearance of crystals characterized by a higher energy content; thehigher is the speed of the particles, the higher the generated energywill be.

In the ultra-micronization process, such technology has been furthermodified, and it provides for:

-   -   an increase of the micronization chamber inner diameter from 200        to 300 mm;    -   an increase of the fluid jet (air) pressure from 7÷8 Bars to        10÷12 Bars;    -   a reduction of the product feeding from 20÷25 Kg/h to 9÷12 Kg/h.

In an embodiment, the palmitoylethanolamide is crystallized in thepresence of a vinyl polymer before the ultra-micronization step. In suchembodiment, the preferred vinyl polymer is polyvinylpyrrolidone. Thecrystallization can occur from various solvents, but the solvent ofchoice is ethanol. In a preferred aspect, the ratio of PEA andpolyvinylpyrrolidone is about 30:1.

The following table I shows the particle size profile ofultra-micronized PEA compared to the particle size profile obtained withthe micronization according to EP 1 207 870 B1.

TABLE I Product A Product B Particle Micronized Ultra-micronized sizepalmitoylethanolamide palmitoylethanolamide >14 microns Traces Absent<10 microns About 96%  100% <6 microns 80% 99.9% <2 microns Notindicated 59.6% <1 microns Not indicated 14.7% <0.6 microns Nonindicated  2.0%

In order to measure the particle size, a laser particle size analyzer(Malvern Mastersizer) with LALLS (Low Angle Laser Light Scattering)technique using the Fraunhofer theory of computation is employed.

Modulated Differential Scanning calorimetry (MDSC) and XRD (X-RayDiffraction) tests have been carried out on the thus-obtained product.

The MDSC technique is a known one, the principles and applications ofwhich are described, for example, in S. R. Rabel et al., Journal ofPharmaceutical and Biomedical Analysis, 21 (1999) 339-345. The testsdescribed in the present patent application have been carried out withan equipment TA DSC Q200.

Such differential calorimetry measurement performed with the MDSCtechnique have shown an outstanding difference between the initialproduct and the ultra-micronized product, which difference consists inthe appearance, in the product subjected to ultra-micronization, of apositive peak of exothermal transition at a temperature between 101° C.and 103° C., which is distinctive of structures with a high energycontent; in the original product, such peak results, on the contrary, tobe negative (see FIGS. 3 and 4). The positive exothermal transition peakhas to be interpreted as the signal of the heat developed by thetransformation of the higher energy content form (formed during theultra-micronization process) into the native crystalline form with lowerenergy content.

The MDSC spectrum analysis of ultra-micronized PEA seems to suggest thatthe high energy crystalline form obtained via the ultra-micronizationprocess of the present invention is substantially stable at roomtemperature, since it is converted back into the original low energyform only at temperatures that are near to the product melting point.The net energy transition which characterizes the positive peak at101-103° C. of FIG. 4 is symptomatic of such stability. Differently,progressive transitions at lower temperatures should have to be noticed.

The X-ray diffraction measurements performed with the XRD technique (anXPERT-PRO equipment has been used) with the aim of investigating solidstate of the products, show a particularly significant differencebetween the diffraction spectra that have been obtained; the spectrum ofthe product subjected to ultra-micronization confirms the presence of adifferent crystalline structure compared to the original product (seeFIGS. 5 and 6).

The positions and intensity of the individual peaks in the two productsare reported herein below.

XRD Peaks of original PEA Peak [2-Theta(°)] Relative intensity (counts)4.150 23205 6.189 40155 8.227 393015 10.266 21864 12.305 211389 14.3449087 18.455 141011 20.527 38771 22.599 115799 26.760 6111 28.866 2690630.971 4861 33.094 27098 39.511 35388 43.872 24259 46.062 6122

XRD Peaks of PEA ultramicronized Peak [2-Theta(°)] Relative intensity(counts) 6.155 1396 8.194 8191 12.271 3809 18.438 2524 20.844 1858521.780 23452 22.532 17160 24.003 5214 25.256 5429 31.289 915 36.770 142238.759 1138

Biological Section

Biochemical Tests

Dose measurements in blood have been carried out of2-arachidonoyl-glycerol (2-AG), an endocannabinoid of great importancein the modulation of the activation of cells capable of expressing thecannabinoid CB2receptor; this is the case of many cells belonging to theimmune system as the microglia.

The tests have been carried out in Beagle dogs by administering, underfasting conditions, an aqueous suspension of palmitoylethanolamide in0.5% carboxymethyl cellulose; the animal was administered, in singleadministration, 15 mg/Kg of micronized palmitoylethanolamide andultra-micronized palmitoylethanolamide, respectively. Blood samples attime 0 (immediately before the administration of palmitoylethanolamide)and at times 1 h, 2 h, 3 h have been taken; the blood was centrifugedand immediately frozen at −80° C.

The dose measurements of palmitoylethanolamide (PEA) and2-arachidonoylglycerol (2-AG) have been performed with mass spectrometrymethod as described in Darmani et al., Neuropharmacology (2005); 48:1154-1163.

The data are reported in table II.

TABLE II Concentration in serum in pmol/mL Administered Time 0 Time 1 hTime 2 h Time 3 h treatment PEA 2AG PEA 2AG PEA 2AG PEA 2AG Micronized12.4 1.0 22.2 1.2 15.6 1.6 13.4 1.7 palmitoyleth- anolamide (Group A - 6animals) Ultra- 12.4 1.0 22.4 3.6 16.2 4.2 14.1 4.7 micronizedpalmitoyleth- anolamide (Group B - 6 animals)

Surprisingly, the present inventors have found that the administrationper os of palmitoylethanolamide in the ultra-micronized formdeterminates a quick and huge increase of 2-AG in blood (which increaseis higher than 400% compared to the basal levels). Such increase resultsto be vastly higher compared to that obtained, under identicalconditions, with the administration of micronized palmitoylethanolamide(increase of about 70%). Without being bond to any theory, theobservation that the passage kinetics of palmitoylethanolamide in theblood, following administration, results to be substantially identicalbetween the micronized form and the ultra-micronized one may induce tothink that the huge increase of 2-AG, following the administration ofthe ultra-micronized palmitoylethanolamide, depends on the increasedsynthesis of 2-AG at the level of the nervous structures protected byblood-brain barrier and/or blood-spinal cord barrier, induced by theadministered product. In fact, it is known that the biosynthesis of 2-AGoccurs—on demand—mainly at the level of central nervous structures suchas spinal cord and brain.

The inventors have hypothesized that such increase can be responsible orco-responsible for the pharmacological effects then observed at thelevel of the spinal cord, after administration of ultra-micronizedpalmitoylethanolamide, as it will be detailed in the following of thepresent description.

Pharmacological Activity

Chronic Inflammation of the Peripheral Nerve with Occurrence ofNeuropathic Pain

After sciatic nerve ligation—CCI (carried out as described by Costa etal., Pain 2008; 139:541-550), a series of altered spinal parameters havebeen assessed in the mouse, following peripheral damage, and related tothe activation of microglial cells induced by the peripheral chronicdistress. In particular, TNF-alpha, NGF, NF-kB according to the methoddescribed by Costa et al. (see supra), and IL-1 alpha according to themethod described by Fiorentino et al., 2008; 58(10):3100-3109 have beenmeasured.

Per os treatments have been carried out, by means of tube, using bothmicronized palmitoylethanolamide suspended in vehicle, andultra-micronized palmitoylethanolamide suspended in vehicle; the resultshave been compared to control animals treated with vehicle alone and toanimals with sciatic nerve ligation treated with vehicle alone. A 0.5%solution in carboxymethyl cellulose has been used as a vehicle.

The administration of the vehicle and the two different suspensionscontaining palmitoylethanolamide has been carried out once per day,starting from the day of the sciatic nerve ligation.

The measurements of the parameters indicated above have been carried outat day 10 from the sciatic nerve ligation, after sacrifice of the testanimal and sampling of the spinal area.

The results are reported in table III.

TABLE III Groups of animals (10 Dose measurements at day 10 afterligation (CCI) animals/ TNF-alpha NGF NF-kB IL-1alpha group) (pg/mgprot) (pg/mg prot) (pg/mg prot) (pg/mg prot) Sham/ 51.0 ± 2.5 28.6 ± 2.00.49 ± 0.002 0.06 ± 0.0002 vehicle (control) CCI/ 66.2 ± 3.1 44.0 ± 6.20.58 ± 0.003 32.20 ± 2.5   vehicle CCI/ 60.4 ± 3.0 38.6 ± 3.0 0.53 ±0.003 25.80 ± 2.0   micro- nized PEA CCI/ultra- 44.1 ± 2.8 10.2 ± 2.00.44 ± 0.001 2.50 ± 0.02  micro- nized PEA

The data show that the administration of ultra-micronized PEA, unlikemicronized PEA, causes a substantial normalization of all thebiochemical parameters under investigation.

Acute Inflammation of Dermo-Epidermal Tissue Due to Immunogenic Stimuliin the Dog

Beagle dog spontaneously sensitized to Ascaris suum were used. Theanimals were left under fasting conditions overnight, before the oraladministration of palmitoylethanolamide.

The animals were divided into 2 groups of 6 animals each; the firstgroup (group A) was administered, in the form of oral viscous suspensionof 0.5% carboxymethyl cellulose, 10 mg/Kg of micronizedpalmitoylethanolamide; the second group (group B) was administered 10mg/Kg of ultra-micronized palmitoylethanolamide suspended in the samevehicle.

Before and after the administration of palmitoylethanolamide, acutaneous reaction was induced by intradermal injection in the lateraltoracic region of Asc S1 antigen (100 μg/mL). A 2% solution of EvansBlue in saline was administered endovenously (0.4 mL/Kg) 30 minutesbefore the intradermal injection of Asc S1 antigen, so as to be able tovisualize the dermal reaction area.

The dermal reaction with the Asc S1 antigen was induced in the animalsof both groups, before (time 0), at 1, 2, 4, 8, and 24 hours,respectively, after the administration of palmitoylethanolamide. Thedermal reaction area was measured 10 minutes after the injection of AscS1 antigen.

The data are reported in table IV.

TABLE IV Inhibition produced by palmitoylethanolamide on the cutaneousreaction induced by Ascaris suum (%) Micronized Ultramicronizedpalmitoylethanolamide palmitoylethanolamide Time (10 mg/Kg) (10 mg/Kg)(hours) Average value Average value 0 0 0 1   9.4 ± 2.6 20.8 ± 5.4 2  9.3 ± 4.7 32.4 ± 4.7 4 −2.6 ± 4.2 26.0 ± 4.9 8 −0.8 ± 2.0 15.1 ± 9.624 −0.2 ± 2.0  4.5 ± 6.7

The data show that ultra-micronized PEA causes an inhibition of thecutaneous reaction above 20%, in periods of time ranging between 1 and 4hours after treatment, compared to an almost null inhibition obtainedwith micronized PEA.

Effect of Palmitoylethanolamide on the Chronic Inflammation ofConnective Tissue Due to Occurrence of Carrageenan-Induced Granuloma inthe Rat

The pharmacological model of granuloma induced, in the rat, by theintroduction in the subcutaneous tissue of carrageenan-soaked sponge hasbeen used. The model is described in De Filippis et al., J Cell Mol Med.2009; 13(6):1086-1095.

Palmitoylethanolamide, in micronized and ultra-micronized form, wasadministered to two different groups of, animals, by oral route and asolution of 0.5% carboxymethyl cellulose (vehicle) as a vehicle, bymeans of gastric tube; the third group of animals was administered, withsimilar modes, the vehicle alone. The administrations were carried outat time 0 (immediately before the introduction of the sponges), andevery 12 hours for 3 consecutive days. The unit doses administered were10 mg/Kg.

The biochemical parameters relative to pro-algogenic mediators weredetected, after sacrifice of the animal occurred after 96 hours from theintroduction of the sponges, both in the granulomatous tissue(expression of the protein NGF), and at the level of the dorsal rootganglia (RDG) (expression of the protein TNF-alpha and the protein NGF).

The data are reported in table V.

TABLE V Dose measurements in the Dose measurements in the granulomatousdorsal root ganglia tissue Expression Expression of of proteinExpression of Groups of animals protein NGF NGF protein TNF-alpha (10animals/group) (OD = mm²) (OD = mm²) (OD = mm²) Carrageenan + 23.3 ± 2.253.3 ± 3.6 196.1 ± 13.2 vehicle Carrageenan + 14.6 ± 2.1 50.8 ± 3.0151.1 ± 9.6  micronized PEA Carrageenan + ultra-  6.1 ± 1.6 43.8 ± 3.1 96.6 ± 10.4 micronized PEA

In this case also, ultra-micronized PEA causes a much more markeddecrease of NGF levels compared to the micronized PEA.

Effect of Palmitoylethanolamide on the Acute and Chronic IntestinalInflammation in the Mouse

It has been recently shown that abnormalities of the enteric nervoussystem such as neuronal degeneration and the decrease in the number ofthe enteric neurons represent critical elements in the pathogeneticmechanism of gastrointestinal disorders such as the Irritable BowelSyndrome.

An acute inflammation was induced in the animal by means ofintra-peritoneal injection of LPS (lipopolysaccharide): the animals weresacrificed after 18 hours from the LPS administration. Instead, achronic inflammation at the colon level was induced by administering theanimal with DNBS (2,4-Dinitrobenzene sulfonic acid): in this case, theanimals were sacrificed after 96 hours from the DNBS administration.

In the acute model, palmitoylethanolamide was administered 15 minutesbefore and 2 hours after the LPS administration. In the chronic model,instead, the palmitoylethanolamide was administered daily for 96 hoursafter the DNBS administration.

The level of TNF-alpha and the percent variation in the number of mastcells were assessed on intestinal tissue.

The results are reported in table VI.

TABLE VI DNBS-induced chronic LPS-induced acute inflammationinflammation Percent Percent Expression decrease Groups of Expressiondecrease in of protein in the animals of protein the number TNF- numberof (10 animals/ TNF-alpha of tissue alpha tissue group) (OD = mm²) mastcells (OD = mm²) mast cells Vehicle 142.1 ± 11.2 100 195.1 ± 14.8 100Micronized 128.3 ± 10.8 86 134.8 ± 8.4  74 PEA Ultra-  75.6 ± 12.3 41 62.9 ± 11.8 26 micronized PEA

Also this model highlights an activity that is much more marked forultra-micronized PEA than for micronized PEA.

In Vivo Effect of Palmitoylethanolamide on the Beta-Amyloid-InducedNeuroinflammation in Mouse

It has been shown how the in vivo administration of beta-amyloid in themouse induces a reactive gliosis with manifestations corresponding tothe ones that are pointed out in the Alzheimer's disease.

C57BL/6 mice with age ranging between 3 and 5 months have been used,divided in 3 different groups (20 animals per group). Two groups wereadministered, per os by means of a tube, with micronized andultra-micronized palmitoylethanolamide, respectively, with a 0.5%solution of carboxymethyl cellulose as a vechicle. The administrationswere carried out daily for 8 days successive to the inoculation of thebeta-amyloid. The third group was administered with the vehicle alone.After sacrifice of the animal, IL-1 beta and NO₂ dose level was measuredin the hippocampal homogenate via an immunofluorescence method.

The experimental method described by Esposito et al., Br J Pharmacol.2007; 151:1272-1279 has been used.

The data are reported in table VII.

TABLE VII Hippocampal tissue levels of IL-1 beta (with Hippocampallevels immunofluorescence method) of NO₂ (in μM/μg (count of cells ofproteins from Groups of animals immunopositive to THE-1 hippocampal (10animals/group) beta) homogeneate) Beta amyloid + 96.1 ± 7.3 9.8 ± 2.3vehicle Beta amyloid + 86.3 ± 7.9 8.2 ± 2.1 micronized PEA Betaamyloid + 21.8 ± 5.4 3.1 ± 0.6 ultra-micronized PEA

The ultra-micronized PEA highlights, in this case also, a high decreaseof the levels of the biological parameters considered, while themicronized PEA shows only a marginal activity.

Clinical Results

Effect of Palmitoylethanolamide on the Control of the PeripheralNeuropathic Pain in Subjects Affected by Multiple Sclerosis

In order to assess the effect of Palmitoylethanolamide on the control ofthe neuropathic pain in Multiple Sclerosis, micronized andultra-micronized palmitoylethanolamide, respectively, in the form oftablets having identical composition in excipients, was administered totwo groups of patients, suitably randomized (10 patients for group), allbeing affected by Multiple Sclerosis, having neuropathic pain at thelower limbs (Central Pain Dyndrome), characterized by dysesthesia,allodynia, paresthesias, cramp-likepains, and foot burning feeling; thedosing used was 600 mg per day for 60 days. The pain intensity wasmeasured with a VAS (Visual Analogue Scale) scale both before and at theend of the treatment with palmitoylethanolamide.

A marked decrease of the pain is evidenced in patients treated withultra-micronized PEA. A statistical analysis has been carried out withthe Wilcoxon test for paired data; the obtained results show a highstatistical significance (p=0.001).

The data are reported in table VIII.

TABLE VIII VAS before the VAS after the treatment Groups of patientstreatment of (10 patients/group) (average value) 60 days (average value)Micronized 6.52 4.22 palmitoylethanolamide Ultra-micronized 6.52 2.85palmitoylethanolamide

Therefore, it is an object of the present invention a pharmaceuticalformulation for human or veterinary use, containing ultra-micronizedpalmitoylethanolamide, as defined above, together with apharmaceutically acceptable excipient.

In an embodiment, more than 99% by weight, or about 99.9% by weight, ofpalmitoylethanolamide has particle sizes lower than 6 microns.

In an embodiment, between 55% and 65% by weight, or between 59% and 60%by weight, of palmitoylethanolamide has particle sizes lower than 2microns.

In an embodiment, between 13% and 17% by weight, or between 14% and 15%by weight, of palmitoylethanolamide has particle sizes lower than 1micron.

In an embodiment, between 1% and 3% by weight, or about 2% by weight, ofpalmitoylethanolamide has particle sizes lower than 0.6 microns.

The formulation according to the present invention can be suitable for aoral, buccal, parenteral, rectal, or transdermal administration, or itcan exist in a form that is suitable for the administration byinhalation or insufflation (both per os and via the nasal route).

For the oral administration, the pharmaceutical compositions can be, forexample, in the form of tablets or capsules that are prepared in theconventional manner, with pharmaceutically acceptable excipients such asbinding agents (for example, pregelatinized corn starch,polyvinylpyrrolidone, or hydroxypropyl methyl cellulose); filling agents(for example, lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (for example, magnesium stearate, talc, orsilica); disgregating agents (for example, potato starch, or sodiumstarch glycolate); or imbibing agents (for example, sodium laurylsulphate). The tablets may be coated with the methods well known in theart. The liquid preparations for oral administration can be, forexample, in the forms of solutions, syrups, or suspensions, or they canbe in the form of freeze-dried products to be reconstituted, before use,with water or other suitable vehicles. Such liquid preparations can beprepared through the conventional methods with the pharmaceuticallyacceptable additives, such as suspending agents (for example, sorbitolsyrup, cellulose derivatives, or edible hydrogenated fats);emulsionating agents (for example, lecithin or acacia); non-aqueousvehicles (for example, almond oil, oily esters, ethyl alcohol orfractionated vegetal oils); and preservatives (for example, methyl- orpropyl-p-hydroxybenzoates or sorbic acid). The preparation can alsosuitably contain aromas, colorants and sweetening agents.

The preparations for oral administration can be formulated in a suitableway to allow the controlled release of the active principle.

For the buccal administration, the compositions can be in the form oftablets that are formulated in the conventional manner, suitable for anabsorption at the level of the buccal mucose. Typical buccalformulations are the tablets for sub-lingual administration.

The formulations of the present invention can be adapted for aparenteral administration by injection. The formulations for theinjections can be presented in the form of a single dose, for example,in ampoules, with the addition of a preservative. The compositions canbe in such a form as suspensions, solutions, or emulsions in oily oraqueous vehicles, and may contain prescribed agents, such as suspendingagents, stabilizers, and/or dispersants. Alternatively, the activeprinciple can be in the form of powder to be reconstituted, before theuse, with a suitable vehicle, for example, with sterile water.

According to the present invention, the compound can also be formulatedaccording to rectal compositions, such as suppositories or retentionenema, for example, containing the base components of the typicalsuppositories, such as cocoa butter or other glycerides.

In addition to the compositions described before, the PEA can also beformulated as a depot preparation. Such long-acting formulations can beadministered via implant (for example, subcutaneously, transcutaneously,or intramuscularly), or by intramuscular injection. Therefore, forexample, it can contain appropriate polymeric or hydrophobic materials(for example, in the form of an emulsion in a suitable oil) or ionicexchange resins, or as minimally soluble derivatives, for example, as aminimally soluble salt.

According to the present invention, the dose of palmitoylethanolamideproposed for the administration to a man (having a body weight of about70 Kg) ranges from 0.1 mg to 2 g, and preferably from 50 mg to 1000 mgof the active principle per dose unit. The dose unit can beadministered, for example, from 1 to 4 times per day. The dose willdepend on the route of administration selected. It shall be consideredthat it could be necessary to make continuous variations of the dosingaccording to the patient's age and weight, and also to the severity ofthe clinical condition to be treated. The exact dose and route ofadministration will be ultimately at the discretion of the attendingphysician or veterinary.

In an embodiment, the ultra-micronized PEA is used in combination withanti-oxidant substances, preferably selected from the group consistingin Quercetin, Resveratrol, Polydatin, Luteolin, Tocopherol, and ThiocticAcid in a therapeutically effective amount.

Examples of formulations containing ultra-micronized PEA (PEA UM)according to the invention are reported herein below.

FORMULATION EXAMPLES Example 1

Each tablet contains:

UM PEA mg 300.00 Microcrystalline cellulose mg 78.47 Sodiumcrosscaramellose mg 45.00 Polyvinylpyrrolidone mg 10.00 Magnesiumstearate mg 4.00 Polysorbate 80 mg 2.00

Example 2

Each tablet contains:

UM PEA mg 600.00 Microcrystalline cellulose mg 156.94 Sodiumcrosscaramellose mg 90.00 Polyvinylpyrrolidone mg 20.00 Magnesiumstearate mg 8.00 Polysorbate 80 mg 4.00

Example 3

Each bilayer tablet contains:

Layer a UM PEA mg 400.00 Pharmacologically acceptable excipients mg200.00 Layer b Trans-Polydatin mg 40.00 Pharmacologically acceptableexcipients mg 25.00

Example 4

Each bilayer tablet contains:

Layer a UM PEA mg 600.00 Pharmacologically acceptable excipients mg280.00 Layer b Luteolin mg 80.00 Pharmacologically acceptable excipientsmg 46.00

Example 5

Each three-layer tablet contains:

Layer a Hyaluronic acid, sodium salt mg 20.00 Pharmacologicallyacceptable excipients mg 15.00 Layer b UM PEA mg 300.00Pharmacologically acceptable excipients mg 152.00 Layer c Hyaluronicacid, sodium salt mg 20.00 Pharmacologically acceptable excipients mg15.00

Example 6

A 5 g dose of orally disintegrating microgranules, for pediatric use,contains:

UM PEA mg 50.00 Non-cariogenic sugar mg 200.00 Pharmacologicallyacceptable excipients q.s. to g 5.00

Example 7

A 5 mL dose of sterile suspension, for pediatric use, contains:

UM PEA mg 80.00 Carboxymethyl cellulose mg 25.00 Bidistilled water q.s.to mL 5.00

Example 8

A 5 g dose of orally disintegrating microgranules contains:

UM PEA mg 600.00 Luteolin mg 100.00 Non-cariogenic sugar mg 200.00Pharmacologically acceptable excipients q.s. to g 5.00

Example 9

Each 5 mL sterile monodose bilayer container contains:

In the aqueous gel: Hyaluronic acid, sodium salt mg 80.00 Bidistilledwater, q.b. to mL 2.50 In the oily gel: UM PEA mg 600.00 Glycerylmonostearate (Geleol) mg 40.00 Vegetal oil q.s. to mL 2.50

Example 10

Each 100 mL sterile bottle for intraperitoneal application contains:

UM PEA g 2.00 Hyaluronic acid, sodium salt g 2.00 Bidistilled water q.s.to mL 100.00

Example 11

Each soft gelatin capsule, for veterinary use (dog and cat), contains:

UM PEA mg 100.00 Phosphatidylserine mg 50.00 Resveratrol mg 60.00Pharmaceutically acceptable oily excipients mg 300.00

The above-described formulations can be prepared according to methodsthat are well known to those skilled in the art, such as those describedin Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co., N.Y.,USA, 17th edition, 1985.

The PEA is a commercial compound, or it can be anyway prepared accordingto methods that are well known to those skilled in the art.

The formulations of the present invention can be used for the treatmentor prophylaxis of neuroimmunogenic inflammatory diseases at the level ofperipheral organs and/or neuroinflammatory diseases, also associated toneurodegeneration at the level of the spinal cord and/or brain.

In particular, the present invention relates to the above-describedformulations for the treatment of:

1—neuroimmunogenic inflammatory processes at the level of peripheralorgans and apparatuses of the body, supporting diseases such as a) theIrritable Bowel Syndrome; b) the interstitial cistitis and the recurrentcistites; c) the vulvodynias and the vestibulodynias; d) the vulvarvestibulitis; e) the endometrial lesions; f) the miastenia gravis g) thechronic abacterial prostatitis of type IIIA and IIIB; h) thearthropathies of traumatic or degenerative or immunologic originaffecting the mobile and/or semi-mobile joints; i) the painful diseasesof the intervertebral discs due to neo-innervation andneo-vascolarization of the cartilaginous tissue and the annexedligamentous structures—pulpy nucleus (nucleus pulposus) and/or fibrousrings (anulus fibrosus), anterior and posterior longitudinal ligaments,supraspinous ligament—; l) the cephalalgic syndromes due to inflammationof the meningeal tissue; m) the inflammations of the mucous andmucocutaneous tissues of the oral cavity and the dental pulp; n) therecurrent fevers on auto-inflammatory basis of PFAPA type in thepediatric age; o) the dermo-epidermal neuralgias of the small fibres,nociceptive and/or pruriceptive, with a neuropathic basis as thepostherpetic neuralgia, the diabetes-associated neuralgias, theneuralgia due to HIV infection, the neuropathic and/or psicogenicitches; p) the granulomas affecting the dermo-epidermal tissue; q) theadherential syndromes due to peritonitis and/or laparotomic and/orlaparoscopic surgical events; r) the dermatologic diseases, also withimmunological genesis, characterized by neuroinflammatory processes,both acute and chronic;

2—Neuroinflammatory processes, also associated to neurodegeneration,that occur and affect nervous structures of the spinal cord following:a) traumatic, dismetabolic, or degenerative noxae such as the medullarycanal stenoses, such as the spondylosis and the spondylolisthesis or thetraumatic lesions from flexo-extension of the spine; b) inflammatorydistresses affecting encephalic nervous structures (stroke, multiplesclerosis, Parkinson's disease, fibromyalgic syndrome) with consequentoccurrence of peripheral pains, currently classified as “Central PainSyndromes”; c) chronic inflammatory distresses of the OsteoarticularSystem and the Peripheral Nervous System, mainly characterized bychronic and/or neuropathic pain;

3—neuroinflammatory processes, also associated to neurodegeneration,that occur and affect nervous structures of given encephalic areasfollowing traumatic, neuro-toxic, dismetabolic, or degenerative noxae,such as the hypoxic distress states (stroke, TIA-Trans Ischemic Attack),the senile and presenile dementias also of the Alzheimer type,cranio-encephalic traumas, Parkinson's disease, Multiple Sclerosis,Amiotrophic Lateral Sclerosis.

As described before, without being bond to a particular theory, it seemsthat such pharmacological effect is mediated by the ability ofultra-micronized PEA to significantly increase the release of theendocannabinoid 2-arachidonoylglycerol (2-AG).

Therefore, it is a further object of the invention a formulationcontaining ultra-micronized PEA as defined above, for the treatment orprophylaxis of neuroimmunogenic inflammatory diseases at the level ofperipheral organs, and/or neuroinflammatory diseases, also associated toneurodegeneration at the level of the spinal cord and/or brain, so as toobtain in the serum of the treated subject, in a period of time between1 and 3 hours after said treatment, concentrations of2-arachidonoylglycerol that are higher, preferably from 3 to 5 foldhigher, than the concentrations before the treatment.

It shall be apparent that, to the present invention, one of ordinaryskill in the art, with the aim of meeting contingent and specific needs,will be able to make further modifications and variations, all of whichare within the protection scope of the invention, as defined by thefollowing claims.

1. A pharmaceutical composition for human or veterinary use, containinga therapeutically effective amount of palmitoylethanolamide in theultra-micronized form, wherein more than 90% by weight ofpalmitoylethanolamide has particle sizes lower than 6 microns, togetherwith pharmaceutically acceptable excipients.
 2. The compositionaccording to claim 1, wherein more than 99% by weight, or about 99.9% byweight, of palmitoylethanolamide has particle sizes lower than 6microns.
 3. The composition according to claim 1, wherein between 55%and 65% by weight, or between 59% and 60% by weight, ofpalmitoylethanolamide has particle sizes lower than 2 microns.
 4. Thecomposition according to claim 1, wherein between 13% and 17% by weight,or between 14% and 15% by weight, of palmitoylethanolamide has particlesizes lower than 1 microns.
 5. The composition according to claim 1,wherein between 1% and 3% by weight, or about 2% by weight, ofpalmitoylethanolamide has particle sizes lower than 0.6 microns.
 6. Thecomposition according to claim 1, wherein said palmitoylethanolamide hasa MDSC spectrum with exothermal transition at temperatures rangingbetween 101° C. and 103° C.
 7. The composition according to claim 1,wherein said palmitoylethanolamide has a spectrum XRD as reported in thefollowing table: Peak [2-Theta (°)] 6.155 8.194 12.271 18.438 20.84421.780 22.532 24.003 25.256 31.289 36.770 38.759


8. The composition according to claim 1, wherein saidpalmitoylethanolamide is in combination with an antioxidant compound. 9.The composition according to claim 8, wherein said antioxidant isselected from Quercetin, Resveratrol, Polydatin, Luteolin, Tocopherol,and Thioctic Acid.
 10. A method of treatment or prophylaxis ofneuroimmunogenic inflammatory diseases at the level of peripheralorgans, and/or neuroinflammatory diseases, also associated toneurodegeneration at the level of the spinal cord and/or brain, themethod comprising administering to a patient in need thereof atherapeutically effective amount of palmitoylethanolamide in theultra-micronized form, wherein more than 90% by weight ofpalmitoylethanolamide has particle sizes lower than 6 microns.
 11. Themethod according to claim 10, wherein said diseases comprise:neuroimmunogenic inflammatory processes at the level of peripheralorgans and apparatuses of the body, supporting diseases such as a) theIrritable Bowel Syndrome; b) the interstitial cistitis and the recurrentcistites; c) the vulvodynias and the vestibulodynias; d) the vulvarvestibulitis; e) the endometrial lesions; f) the miastenia gravis g) thechronic abacterial prostatitis of type IIIA and IIIB; h) thearthropathies of traumatic or degenerative or immunologic origin,affecting the mobile and/or semi-mobile joints; i) the painful diseasesof the intervertebral discs due to neo-innervation andneo-vascolarization of the cartilaginous tissue and the annexedligamentous structures—pulpy nucleus (nucleus pulposus) and/or fibrousrings (anulus fibrosus), anterior and posterior longitudinal ligaments,supraspinous ligament—; l) the cephalalgic syndromes due toinflammation, of the meningeal tissue; m) the inflammations of themucous and mucocutaneous tissues of the oral cavity and the dental pulp;n) the recurrent fevers with auto-inflammatory basis of PFAPA type inthe pediatric age; o) the dermo-epidermal neuralgias of the smallfibres, nociceptive and/or pruriceptive, with neuropathic basis as thepostherpetic neuralgia, the diabetes-associated neuralgias, theneuralgia due to HIV infection, the neuropathic and/or psicogenicitches; p) the granulomas affecting the dermo-epidermal tissue; q) theadherential syndromes due to peritonitis and/or laparotomic and/orlaparoscopic surgical events; r) the dermatologic diseases, also withimmunological genesis, characterized by neuroinflammatory processes,both acute and chronic; neuroinflammatory processes, also associated toneurodegeneration, which occur and affect the nervous structures of thespinal cord following: a) traumatic, dismetabolic, or degenerative noxaesuch as the medullary canal stenoses, such as the spondylosis and thespondylolisthesis or the traumatic lesions from flexo-extension of thespine; b) inflammatory distresses affecting encephalic nervousstructures (stroke, multiple sclerosis, Parkinson's disease,fibromyalgic syndrome) with consequent occurrence of peripheral pains,currently classified as “Central Pain Syndromes”; c) chronicinflammatory distresses of the Osteoarticular System and the PeripheralNervous System, mainly characterized by chronic and/or neuropathic pain;neuroinflammatory processes, also associated to neurodegeneration, whichoccur and affect nervous structures of given encephalic areas followingtraumatic, neuro-toxic, dismetabolic, or degenerative noxae, such as thehypoxic distress states (stroke, TIA-Trans Ischemic Attack), the senileand presenile dementias also of the Alzheimer type, cranio-encephalictraumas, Parkinson's disease, Multiple sclerosis, Amiotrophic LateralSclerosis.
 12. The method according to claim 10, wherein said treatmentis carried out so as to obtain, in the serum of the treated subject, ina period of time between 1 and 3 hours after said treatment,concentrations of 2-arachidonoylglycerol from 3 to 5 fold higher thanthe concentrations before the treatment.
 13. A method for themicronization of palmitoylethanolamide, wherein said method is carriedout in fluid jet plant.
 14. The method according to claim 13, whereinsaid fluid jet plant operates with “spiral technology” with pressurizedair jet.
 15. The method according to claim 13, wherein said methodoperates at a pressure of 10-12 bars of the fluid jet, and with aproduct feeding of 9-12 Kg/h.
 16. The method according to claim 13,wherein said plant comprises a micronization chamber of about 300 mmdiameter.
 17. The method according to claim 13, wherein thepalmitoylethanolamide is crystallized in the presence of a vinyl polymerbefore the ultra-micronization step.
 18. The method according to claim17, wherein said vinyl polymer is polyvinylpyrrolidone.
 19. The methodaccording to claim 17, wherein said crystallization is carried out inethanol.
 20. The method according to claim 17, wherein the ratio betweenN-palmitoylethanolamide and polyvinylpyrrolidone is about 30:1. 21.Ultra-micronized palmitoylethanolamide, as it can be obtained with themethod according to claim
 13. 22. A polymorphic form ofpalmitoylethanolamide having a MDSC spectrum with exothermal transitionat temperatures ranging between 101° C. and 103° C., and a spectrum XRDas reported in the following table: Peak [2-Theta (°)] 6.155 8.19412.271 18.438 20.844 21.780 22.532 24.003 25.256 31.289 36.770 38.759


23. The polymorphic form according to claim 22, wherein more than 90% byweight of palmitoylethanolamide has particle sizes lower than 6 microns,and between 13% and 17% by weight, or between 14% and 15% by weight, hasparticle sizes lower than 1 microns.